Dendritic spines are signaling microcompartments that serve as the primary site of synapse formation in neurons. Actin assembly and myosin 2 contractility play major roles in the maturation of spines from filopodial precursors. Myosin 18A is a myosin 2-like protein expressed from flies to man that lacks motor activity, is sub-stochiometric to myosin 2, and co-assembles with myosin 2 to make mixed filaments (Billington et al., 2015). Myosin 18A is alternatively spliced to create multiple isoforms that contain unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. These observations suggest that myosin 18A serves to recruit proteins to mixed filaments of myosin 2 and myosin 18A. One such protein is the Rac/Cdc42 GEF -PIX, which is known to promote spine maturation by activating the NPFs WAVE and WASp, leading to Arp2/3-dependent actin filament assembly (Zhang et al., 2005; Saneyoshi et al., 2008). Here we show that myosin 18A is highly expressed in cerebellar Purkinje neurons and concentrates in spines along with myosin 2 and F-actin. Myosin 18A spine targeting is driven by both co-assembly with myosin 2 and an actin binding site present in myosin 18As N-terminal extension. miRNA-mediated knockdown of myosin 18A results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A. Importantly, -PIX co-localizes with myosin 18A in spines (but not when its myosin 18A binding site is deleted), and its spine localization is lost upon myosin 18A knockdown. These and other data argue that mixed filaments of myosin 2 and myosin 18A in Purkinje neuron spines form a complex with -PIX that promotes spine maturation by activating Arp2/3-dependent actin filament assembly downstream of -PIXs GEF activity. The actin-based motor myosin Va transports numerous cargos, including the endoplasmic reticulum (ER) in cerebellar Purkinje neurons and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Towards that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of ER transport in Purkinje neurons (PNs) and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it and associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including Guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1s localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo. Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and 50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a 50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.